Bangladesh Med Res Counc Bull 2013; 39: 124-129
Microbial contamination in herbal medicines available in Bangladesh Noor R1, Huda N1, Rahman F1, Bashar T2, Munshi SK1 2
1 Department of Microbiology, Stamford University Bangladesh, Dhaka. Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Dhaka. Email:
[email protected]
Abstract Plants have long been used as herbal medicines in many countries. However, microbial contamination of these medicines may affect human health. Present study was performed to assess the pathogenic proliferation in the locally available commercial herbal oral medicines. The pathogenic load was compared with the microbiological standard given by the British Pharmacopoeia. Out of 85 oral liquid samples, 2 were found to be highly contaminated with a total aerobic bacterial load of 1.24×105 cfu/ml, 10 samples were contaminated with fungi (1.2×104-6.3×104 cfu/ml). Tests for specific pathogens were carried out. One sample showed contamination by coliforms but none of the samples were contaminated by Salmonella spp. and Shigella spp. Among 40 semisolid samples, one showed to be contaminated with bacteria (1.93×105 cfu/g) and 5 samples consisted of fungal load ranging between 1.5×104-2.2×104 cfu/g. The presence of bacteria and fungi in these samples thus suggest the fact that aseptic handling is necessary during processing of oral herbal medicines.
Introduction Herbal medicines include herbs, herbal materials, herbal preparations and finished herbal products. Such medicinal preparations have been used since ancient times to treat a wide range of diseases1-3. However, the medicinal use of herbs went into a rapid decline in the Western countries when more predictable synthetic drugs were made commonly available. In contrast, many developing countries continued to get benefit from the rich knowledge of medical herbalism. For example, Ayurvedic medicine in India, Kampo medicine in Japan, traditional Chinese medicine (TCM), and Unani medicine in the Middle East and South Asia are still being used by a large majority of people4. The World Health Organization (WHO) survey indicated that about 70-80% of the world population particularly in the developing countries rely on non-conventional medicines mainly of herbal origins for their primary health care. This is because herbal medicines are relatively accessible and cheaper than the synthetic drugs5. Many plants are used in traditional medications as herbal preparations for human health-care6 and they are being promoted as natural and safe without any side effects. As the use of herbal preparations by patients is increasing day by day, there is a need for pharmacists and physicians to have knowledge about the safety of these preparations3. The unscientific methods of collection, storage,
transportation and congenial climatic conditions can render the raw materials for herbal drugs prone to fungal infestations. The raw materials collected using unscientific methods are commonly exposed to many pathogenic contaminants and are often deteriorated by pathogenic microorganisms before harvesting, and also during handling and storage6. Therefore, lack of regulation for herbal supplements presents potential health risk, largely their contamination chances with pathogenic microorganisms. However, only a few surveillance studies have been conducted to assess this threat6. Present study figured out the biological threats in herbal medicines and added the knowledge of proliferating bacteria, yeasts and moulds in such medicines. Evidence of human contamination of herbs has also been reported7. Microbial contamination is assumed to occur through handling by personnel who are infected with pathogenic bacteria during harvesting/collection, post-harvest processing and the drug manufacturing process. This should be controlled by implementing best practice guidelines such as Good Manufacturing Practice (GMP)8. The microbiological quality of medicinal products became noteworthy in 1966 when over 200 cases of salmonellosis were reported from consumption of contaminated thyroid tablets, demonstrating that microbial contamination of medications can result
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in clinical infection9. Fungal contamination has been reported to affect the chemical composition of the raw materials and thereby decrease the medicinal potency of herbal drugs6. The most prominent fungal toxins reported are aflatoxins, zearalenone, ochratoxin and patulin, which are collectively known to cause hazards to the liver, nervous system, muscular system, respiratory organs as well as digestive and genital systems6. Based on this fact, we demonstrated the bacterial and fungal population in the locally available herbal medicines. According to the WHO technical guidelines for the assessment of microbial quality of herbal preparations, determination of microbiological contaminants and limit tests for total viable aerobic bacteria and fungi indicate the quality of herbal preparations. According to the British Pharmacopoeia (2004) standards, Salmonella and Shigella species must not be present in herbal medicines intended for internal use, at any stage10. Other microorganisms should be tested and should comply with limits set in regional, national or international pharmacopoeias8. Considering the total contamination chances and based on the recommended guidelines or standards, present study was designed to assess the microbiological contamination in commercial herbal oral medicines and to compare the pathogenic load with the microbiological standards for herbal preparations given by the British Pharmacopoeia. The objectives of the study were: 1) to determine the nature of microbial contamination of the herbal medicines, 2) to determine the extent of such contamination, 3) to compare the results of this study with the standards set by British Pharmacopoeia.
Materials and Methods All materials and chemical reagents were of analytical grade. Experiments were done carefully with appropriate controls. Settings This cross sectional study was carried out on locally available herbal medicines from November, 2010 to November, 2011 in the Department of Microbiology, Stamford University Bangladesh. Sampling One hundred twenty five (125) samples of Unani and Ayurvedic preparations were collected from retailers at Sonargaon thana of Narayangonj District, Bangladesh, for assessment of their microbial contamination. Eighty five (85) oral liquid samples from 16 different batches and 40
oral semisolid products of 19 different batches were tested for microbial limits. Coliforms and two specific pathogens Salmonella spp. and Shigella spp. were checked for their presence along with the total aerobic bacterial count as well as the combined yeast and mould count. Table I shows the relevant sample codes with the number of samples of herbal oral liquids and semisolids. Samples of herbal medicines were collected in sterile glass beaker and special care was taken to prevent accidental contamination of the samples during its collection and transportation to the testing laboratory. Pre-Treatment of the Sample Sterile inactivating agent (Polysorbate 80) was added aseptically to the diluents (Nutrient broth) as the samples had antimicrobial activity and was mixed properly. Ten gram or 10 ml of sample was aseptically transferred in a 150 ml sterile conical flask. A 10-fold dilution was prepared by mixing 10g or10 ml of sample with diluents and was mixed properly. Serial dilutions (up to 10-3) were made based on the expected level of microorganisms in the sample being examined. Assessment of Pathogens Methods prescribed in the British Pharmacopoeia (2004) were used to test microbial quality of the herbal medicines10. In this general method, certain selective media were used. A feature common to all selective media was that the sub-lethally injured organisms could not be detected. Soybean-Casein Digest agar media were used to enumerate the total bacterial population. Sabouraud Dextrose agar (SDA) was used for the identification and enumeration of total fungi. Xylose-Lysine Deoxycholate (XLD) agar for the isolation of Salmonella spp. and Shigella spp., and MacConkey (MAC) agar media for isolation of coliforms were used. The condition of the test for microbial contamination was designed to minimize accidental contamination of the material being examined. The precautions taken during the study did not affect any microorganism10. Standard Organisms Used for Comparison Escherichia coli ATCC 8739, Salmonella typhimurium ATCC 13311, Aspergillus brasiliensis ATCC 16404 were used for the growth promotion test during the estimation of total aerobic bacterial count. They were also used as positive control during isolation and identification of coliforms, Salmonella species, yeasts and moulds. Geobacillus stearothermophilus ATCC 7953 (Spore strip) were used for the validation of autoclave sterilization.
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Total Aerobic Count (for Bacteria and Fungi) Spread plate technique was performed for the detection of total aerobic count (bacteria, yeast & mould). One ml of the treated sample from the required dilution was spread over the culture media and incubated at 35 °C - 37 °C for 48 to 72 hours for allowing the growth of aerobic bacteria and 22 °C - 25 °C for 5 to 7 days for the growth promotion of total combined yeast and mould. After incubation, the individual colonies were counted. The arithmetic averages of the counts were taken and the number of colony forming units (cfu) was calculated per gram or milliliter. Selective media were used to detect specific pathogens by streak plate method. Triplicate experiments were carried out and reproducible results were found. Identification of Coliforms, Salmonella spp. and Shigella spp. Enrichment was done to enhance the growth of pathogenic bacteria. Ten gram or 10 ml of sample was aseptically transferred in a 150 ml sterile conical flask marked with sample code. The sample was mixed with sufficient amount of lactose broth medium to make the final volume reaching to 100 ml and was mixed properly following incubation at 35 °C - 37 °C for 18 to 24 hours for bacterial enrichment. One loop full of enriched sample was streaked aseptically by using a sterile inoculating loop on the surface of MacConkey agar and Xylose-Lysine Deoxycholate (XLD) agar media. The media were incubated at 35 °C - 37 °C for 18 to 24 hours. Then the plates were observed for isolated characteristic colonies. The characteristic lactose fermenting pink colonies rather than nonlactose fermenter ones were detected and picked from MacConkey agar and Eosine Methylene Blue (EMB) agar was used for confirmation of coliforms. Triple Sugar Iron (TSI) agar test was also used for the biochemical identification of the isolates. The presence of coliforms was confirmed by the appearance of bluish-black colonies with green metallic sheen on EMB agar medium.
Results Quantification of Pathogens and Comparison with the Set Standard Out of 85 liquid samples, 2 samples showed very high bacterial load (1.24×105 cfu/ml), 10 samples showed high yeast & mould (1.2×104 -6.3×104 cfu/ml) growth that did not comply with the given standards (tables I & III). One sample showed contamination by coliforms which are intestinal bacteria and are indicator for contamination by feces. None of the samples showed contamination by Salmonella spp and Shigella spp. Out of 40
semisolid samples, one sample showed very high bacterial load (1.93×105 cfu/g), 5 samples showed high yeast & mould (1.5×104-2.2×104 cfu/g) growth that did not comply with the given standards (Tables II & IV). None of the samples showed contamination by E. coli and Salmonella spp. Bacterial Pathogens in the Herbal Medicines As shown in Table I, sample no. 5 showed the bacterial growth at the highest dilution as too numerous to count (TNTC). Sample code SJer, sample no. one showed the total bacterial count 1.24×105 cfu/ml that did not comply with the given standard. In semisolid products, sample code EZam, sample no. one showed the total bacterial count of 1.93×105 cfu/g that did not comply with the given standard (Table II). However, others complied with the standard given by British Pharmacopoeia. Fungal Pathogens in the Herbal Medicines Among the liquid products, sample code SCar, sample no. 3 & 7 showed that the total yeast & mould count was 2.5×104 cfu/ml and 1.2×105 cfu/ml; code SSaf, sample no. 3 & 8 showed the total count 7.7×104cfu/ml and 3.6×104cfu/ml, respectively (table II). Code SNau, sample no. 3, 5 & 6 consecutively showed the total count 2×104 cfu/ml, TNTC at the highest dilution and 6.3×104 cfu/ml; code SIct, sample no. 3 showed that the total count was 1.8×104cfu/ml; code SFev, sample no. one showed the total count 1.2×104 cfu/ml and sample code SJer and the sample no. one consisted of 2.2×105 cfu/ml of total fungi (Table III), which did not comply with the given standard. Presence of pathogens could pose serious health risk as discussed later. In semisolid products, sample code MUsh, sample no. 1, 3 & 4 consecutively showed the total yeast & mould count 2.2×104cfu/g, 1.7×104cfu/g and 2×104cfu/g (table IV). Code MMum, sample no. one showed the total count 1.5×104cfu/g; code EZam, sample no. one consisted of 1.5×104 cfu/g (Table IV) of aerobic pathogens that did not comply with the given standard of British Pharmacopoeia. All the other samples complied with the given standard. Specific Pathogens in the Herbal Medicines One liquid product, sample code SNau, sample no. 6 showed the presence of coliforms that did not comply with the given standard. All other samples complied with the standard for coliforms as given in British Pharmacopoeia. All of the samples were free of Salmonella spp. and Shigella spp., and hence complied with the given standard.
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Table I: Determination of total viable aerobic bacterial count in oral liquid samples Sl. Sample No. of Total number of viable aerobic bacterial count (cfu/ml or cfu/g) No. Code Samples S–1 S–2 S–3 S–4 S–5 S–6 S–7 S–8 S–9 2 01 SCin 10
